Issued January 17, J9J2. 

U. S. DEPARTMENT OF AGRICULTURE, 

BUREAU OF ANIMAL INDUSTRY,— Bulletin 145. 

A. D. MELVIN, Chief of Bureau, 



TRYPANOSOMA AMERICANUM, 

A COMMON BLOOD PARASITE OF AMERICAN CATTLE. 



BY 

HOWARD CRAWLHY, 

Junior Zoologist^ Zoological Division. 




WASHINGTON: 

GOVERNMENT PRINTING OFFICE. 

1912. 




Class 'r>r^Q-| 

Rnnk C ? .? 



X 



7 



Issued January 17, 1912. 

U. S. DEPARTMENT OF AGRICULTURE, ^yg< 

BUREAU OF ANIMAL INDUSTRY.— Bulletin 145. 



A. D. MELVIN, Chief of Bureau. 



TRYPANOSOMA AMERICANUM, 

A COMMON BLOOD PARASITE OF AMERICAN CATTLE. 



BY 



HOWARD CRAWLEY. 

Junior Zoologtsl, Zoological Division. 




WASHINGTON: 

GOVERNMENT PRINTING OFFICE. 

1912. 



Md 



^ 






THE BUREAU OF ANIMAL INDUSTRY. 



Chief: A. D. Melvin. 
Assistant Chief: A. M. Farrington. 
Chief Ckrk: Charles C. Carroll. 

Animal Husbandry Division: George M. Rommel, chief. 
Biochemic Division: M. Dorset, chief. 
Dairy Division: B. H. Rawl, chief. 

Inspection Division: Rice P. Steddom, chief; Morris Wooden, R. A. Ramsat, 
and Albert E. Behnke, associate chiefs. 

Pathological Division: John R. Mohler, chief. 
Quarantine Division: Richard W. Hickman, chief. 
Zoological Division: B. H. Ransom, chief. 
Experiment Station: E. C. Schroeder, superintendent. 
Editor: James M. Pickens. 

ZOOLOGICAL DIVISION. 

Chief: B. H. Ransom. 

Assistant Zoologists: Albert Hassall, Harry W. Graybill and Maurice C. 
Hall. 

Junior Zoologists: Howard Crawley and Winthrop D. Foster. 
2 






LETTER OF TRANSMITTAL. 



U. S. Department of agriculture, 

Bureau of Animal Industry, 

Washington, D. C, August 31, 1911. 
Sir: I have the honor to transmit, and to recommend for pub- 
Ucation in the bulletin series of this bureau, the accompanjong man- 
uscript entitled '' Trypanosoma americanum, a Common Blood Par- 
asite of American Cattle," by Howard Crawley, of the Zoological 
Division. 

A preliminary description of this trypanosome was prepared in 

1909 by Mr. Crawley and published in Bulletin 119 of this bureau. 

The present paper presents a more extended study of the organism, 

which is shown to be harbored by about 75 per cent of adult cattle. 

Respectfully, 

A. D. Melvin, 
Chief of Bureau. 
Hon. James Wilson, 

Secretary of Agriculture. 

3 



CONTENTS. 



Page. 

Introduction ; 5 

R6sxim6 of literature 5 

Method of experimental work 10 

Examination of cultures 10 

Seasonal variation in number of trypanosomes in blood 13 

Effect on the trypanosomes of keeping blood before cultures were made . . 16 

Results obtained with measured quantities of blood 18 

General results of cultural work 20 

Observations on fresh material 21 

Attempts to discover the trypanosome in freshly drawn blood 23 

Evolution of the trypanosomes in cultiu"e 24 

Movements of trj^panosomes in cultures 29 

The process of multiplication 30 

Motility of the trypanosomes 31 

Observations on fixed material 32 

Morphology of the forms in the blood 33 

Morphology of the forms in culture 34 

Cytology of the forms in culture 35 

Principal characteristics of Trypanosoma americanum 38 

Conclusions 38 

Bibliography 39 



ILLUSTRATIONS. 



[All figures made from camera outlines and all on the same scale.] 

Page. 

Fig. 1 and 2. Forms of Trypanosoma am^icanum from centrifuged blood 32, 33 

3. Form from bouillon culture 96 hours old 34 

4 to 9. Forms from bouillon cultures 112 hours old 35 

10. Form from bouillon culture 72 hours old 36 

11 and 12. Forms from bouillon cultures 6 days old 37 

13 to 15. Forme from bouillon cultures 10 days old 37 

4 



TRYPANOSOMA AMERICANUM, A COMMON BLOOD 
PARASITE OF AMERICAN CAHLE. 



INTRODUCTION. 

In Bulletin 119 of the Bureau of Animal Industry there was de- 
scribed, under the name of Trypanosoma americanum, a flagellated 
organism which appeared in bouillon cultures of the blood of American 
cattle. At the time of writing, although but 7 cows had been tested, 
trypanosomes had appeared in the blood cultures of all of them, thus 
indicating that a large proportion of cattle are infected. 

The study therein described had been made primarily on stained 
smears, and based on what was observed in these it was stated that 
the earhest stage noted was a round or oval body, occurring in clusters, 
and, while it showed a kinetonucleus, it lacked a flagellum. Tliis last 
observation is incorrect, the mistake being due to the fact that in these 
early stages the flagellum fails to stain. A more extended study, 
based partly on fresh material, has shown that the organisms in the 
culture tubes are always flagellated. It is desired to emphasize the 
fact that this observational error was made, since the precise nature 
of the earhest organism to appear in the culture tubes is a matter of 
some moment, bearing as it does on the problem of what is the nature 
of the element present in the circulating blood of the cow. It may 
also be stated here at the outset that the results of this more extended 
study show that this latter undoubtedly is a trypanosome Hke that 
present in the culture tubes, because trypanosomes have been found 
in the blood itseK within a few hours after its removal from the cow. 

RESUM6 OF LITERATTJRE. 

There is a very considerable hterature on the trypanosomes of 
cattle, and a number of species have been described as occurring in 
them. It will not be necessary here, however, to do more than con- 
sider that which bears directly on the problem in hand, that is, which 
treats of trypanosomes presumably similar to Trypanosoma ameri- 
canum J Some of this literature was, reviewed in Bulletin 119, but 
one paper which should have been noted was overlooked, and several 
have since come to hand. It may, however, be advisable again to 

1 References to literatiire are given in the bibliography at the end of this bulletin. 



6 TRYPANOSOMA AMERICANUM, A BLOOD PARASITE OF CATTLE. 

refer to Miyajima's work, since this is the point of departure of all the 
studies on this peculiar trjpanosome. 

The organism found by Miyajima in cultures made from the blood 
of Japanese cattle is apparently the same as that found in the United 
States. Unfortunately Miyajima does not give a detailed description 
of his parasite, and his figures are not particularly clear. They show, 
however, that he was dealing with a trypanosome possessing only a 
very short undulating membrane, and his figure 10, of a diAdsion 
form, shows two granules lying at the same level and in the anterior 
part of the body. From this it may be concluded that the Japanese 
form has the characteristic of a kinetonucleus lying very close to the 
trophonucleus. Further, the length, given as five times the diameter 
of a red blood cell, or say 30 microns, is in general agreement with 
what is found here. 

Martini (1909), working at Manila, repeated Miyajima's work, and 
obtained trypanosomes in bouillon culture tubes inoculated with the 
blood of a calf. He used 2 c. c. of blood to 10 c. c. of bouillon. In 
one case the trypanosomes appeared in 33 hours. As a rule, however, 
the period was from 43 to 48 hours. The tubes were kept at a tem- 
perature of 26 to 27° C* The trypanosomes, while in general about 
the size of Trypanosoma lewisi, showed great variation in length. 
Some were only one and one-half to three times as long as the diameter 
of a red blood cell, but as the cultures aged they became longer, and 
on the fifth and sixth days forms were present 20 to 25 times the 
length of the caK's red blood cell.^ The smallest forms were morpho- 
logically the same as the largest. The anterior end of the flagellum 
showed a httle club-shaped swelling. The kinetonucleus lay trans- 
versely, the trophonucleus longitudinally, and the former was gener- 
ally in front of the latter. The parasites grew indifferently in acid 
or alkaline bouillon. In cultures in acid media, the cytoplasm con- 
tained chromatin granules. A chronological transition was not 
observed, and rudimentary forms were always to be found alongside 
of those fully developed. Attempts to inoculate monkeys failed. 

Commenting on the above, there can be no question that Martini 
was dealing with the same trypanosome as Miyajima, which is also, 
in all probability, the one which appears in cultures made from the 
blood of American cattle. 

Wrublewski (1908)^ describes a trypanosome found in the blood 
of the Lithuanian bison. He remarks that this species of Bos is not a 

1 Cultures made in the Zoological Laboratory of the Bureau of Animal Industry on July 13, 1909, showed 
trypanosomes July 15, the mean temperature of the laboratory for this period being 27.4° C. 

s It is to be regretted that Martini has not furnished us with actual measurements. According to his 
own figures, the calf erythrocytes range in diameter from 4 to 6J microns, and this would make the largest 
specimens say 100 to 150 microns long, clearly a gross exaggeration. His figure 12 shows an animal 40 to 
45 microns long; that of figure 14 is perhaps 50, and these are the largest which he figures. . 

s This paper was missed at the time of writing Bulletin 119. 



RESUME OF LITERATURE. 7 

favorable subject for experimentation and hence his material con- 
sisted of smears taken from dead individuals. The trypanosomes found 
ranged in length from 30 to 50 microns. The posterior end is elon- 
gated, but terminates bluntly. In some, the middle portion is broad 
and rounded (compare figs. 13 and 14, this bulletin). In this broad por- 
tion are to be found the kinetonucleus and the trophonucleus, the for- 
mer in front of the latter. The root of the fiagellum is broad and may 
show a knoblike expansion. The free end of the fiagellum is also 
knobbed. Around the trophonucleus are masses of deeply staining 
granules, and granules may also be found scattered throughout the 
entire body. 

In some cases Wrublewski obtained blood sufficiently fresh to 
show living trypanosomes. They are very energetic, and in hanging 
drops dash through the red cells with extraordinary velocity, hurlmg 
these to one side. The parasite moves with the fiagellum in front. 
The movement is rotatory. 

Vladimiroff and Yakimoff (1908) name this trypanosome Trypano- 
soma wrublewski. 

Of the several bovine trypanosomes described as occurring in the 
circulating blood, this comes by far the closest to T. americanum . 
Wrublewski's figures 3, 4, and 5 would answer for specimens of the 
American species. The most noteworthy distinction is in the mo- 
tihty, T. americanum rarely showing a rapid translatory movementv 
There is no especial reason why the two might not be the same, the 
parasite properly belonging to the bison and transferred from it to 
domesticated cattle by natural means. The fact that the parasite 
is apparently quite abundant in the circulating blood of the bison, 
and very scarce in that of ordinary cattle, is no argument against 
their identity. Moreover, the sluggishness of T. americanum in cul- 
ture tubes is no criterion as to how it may behave in the circulating 
blood, regarding which there is, unfortunately, no information at 
hand. 

Recent studies have shown trypanosomes to be present in a large 
proportion of cattle in Germany, or at least capable of appearing in 
cultures made from their blood. 

Knuth and Rauchbaar (1910) tested 17 adult cattle and 2 calves, 
and found 10 of the adults to be parasitized. Later, out of a lot of 9 
cattle, 6 were found infected. 

KJnuth, Rauchbaar, and Morgenstern (1910) found trypanosomes 
in culture tubes from 7 out of 25 cattle. They note the presence in 
the tubes of developmental forms and of agglomerations. 

Behn (1910 /?), culturing cattle blood in the same manner, found 
flagellated forms on the second day. Furthermore, in smears made 
from cultures 1 to 2 days old, he found round bodies, in many cases 



8 TRYPANOSOMA AMEBIC ANUM, A BLOOD PARASITE OF CATTLE. 

with alveolar protoplasm, wliich stained an intense blue with Giemsa. 
These showed one or more large, round nuclei, variable in size and 
staining red. The bodies varied in size, the largest being nearly as 
large as a leucocyte. Some were free, others inclosed in white blood 
cells. Behn appears to consider that the intracellular forms, growing, 
break open the leucocytes and escape. He bases tliis view on the 
fact that in smears from cultures 2 days old the bodies then had 
attached to them the remnants of disintegrated leucocytes. He pro- 
poses the query as to whether he is dealing with a case of pha; )cy- 
tosis or of an evolution of the parasites within the leucocytes, and 
promises a more detailed study. 

No bodies such as the above have been seen in smears made from 
American cattle, and it is perhaps" possible that Behn was dealing with 
moribund or dead leucocytes. 

The same author (Belin, 1910 a) refers to the seven animals men- 
tioned by Kjiuth, Rauchbaar, and Morgenstern. He states that 
whereas the cultures from these were always positive from the begin- 
ning of August until the end of September, after cool weather set in 
they no longer developed trypanosomes. One of these cows failed 
to show parasites after September 11. But in a smear of her blood 
taken August 8 there was found a single trypanosome, remarkable 
for its unusual breadth. 

Behn gives the following measurements: 

Microns. 

Posterior end to middle of kinetonucleus 13 

Middle of kinetonucleus to posterior end of trophonucleus 4 

Trophonucleus 2 

Anterior end of trophonucleus to anterior end 24 

Length of body 43 

Length of free fiagellum 12 

Total length 55 

Maximum width 12 

The trypanosome had the body sharply bent. The cytoplasm 
showed clear spaces and abundant granules. The granules were al- 
most all of the same size and stained reddish to a blue-violet. Around 
the nucleus the cytoplasm was freer from granules than elsewhere. 
The kinetonucleus stained a black violet, and lay in a clear region. 
The nucleus lay transversely, filling the entire width of the body. 
It stained pale red. 

Continuing his studies Behn (1910 f) inoculated a calf with blood 
from a cow whose blood gave positive cultures, but was negative to 
direct examination. Eleven days later the blood of this calf was 
positive to direct examination. For the first few days the trypano- 
somes seen were of the Trypanosoma frariki type, but later they took 
on the appearance of T. theiler-i. Cultures made from the blood of this 
calf, after the trypanosomes had appeared, were positive. 



besum6 of literature. 9 

Kjiuth (1910) tested by the cultural method the blood of 41 cattle, 
with the following results : 



Total 
number 
tested. 



Positive 

for 

try pan 0- 

somes. 



Adult cattle 

Young cattle... 
Sucking calves. 



Schmitt (1910) also found trypanosomes of the theileri type in a 
Pomeranian cow suffering from Texas fever. These were found in the 
blood, were rare, and were present for 10 days. 

Sergent, E. and E., (1911) made cultures of the blood of 82 cattle 
killed at the abattoir at Algiers. Trypanosomes were obtained nine 
times. Division forms were abundant, and subcultures were made. 
The medium used was bouillon. 

Delanoe (1911), at AKort, made cultures of cattle blood, using 3 c. c. 
of defibrinated blood in 10 c. c. of bouillon. Six out of the ten animals 
tested gave positive results, but not all of the cultures made from 
parasitized animals were positive, indicating the great rarity of the 
trypanosomes in the blood. The elements found in the cultures were 
crithidia-like, averaged 50 microns in total length, and the free tip of 
the flagella was broadened. Chromatoid granules appeared in the 
cytoplasm only in the old cultures. In a subculture in Nicolle's 
medium, there were seen very small forms without flagella, flagel- 
lated elements 35 microns long, and trypanosomes with a posterior 
kinetonucleus. 

Knuth confirms Behn in the respect that, after the onset of cold 
weather, animals having given positive results cease to do so. He 
suggests that this may be due either to the disappearance of the 
insect carriers or to the failure of the trypanosomes to grow in the 
tubes in cold weather. Finally, by private correspondence, Knuth 
has been advised that blood cultures of cattle have been made in a 
number of laboratories in Germany, Denmark, and Sweden, with 
the subsequent finding of trypanosomes. 

Dudukalov and Dudukalova (1910) report the results of an experi- 
mental study on the trypanosome of the cow. The culture media 
used were (a) bouillon; (b) equal parts of bovine blood and 0.8 per 
cent NaCl solution;^ (c) agar. Each tube was inoculated with a 
few drops of blood. After 3 to 4 days there appeared in the tubes 
round forms of the parasite, about the size of leucocytes, in many 
cases occurring in great clusters. On the sixth to seventh days 
elongated bodies appeared among these round bodies, and on the 
tenth to twelfth days there were present great numbers of motile 

» The text says 0.08 NaCl solution, doubtless a printer's error. 
10414°— Bull. 145—12 2 



10 TRYPANOSOMA AMERICANUM, A BLOOD PARASITE OP CATTLE. 

trypanosomes provided with long flagella. After 2 to 3 weeks the 
trypanosomes were present in enormous numbers and were of 
various sizes and forms. They remained ahve in the tubes for 2 to 
4 months. Subinoculations were made and carried to the fourth 
generation. The optimum temperature for the cultures is 10 to 
15° R. 

With reference to the above papers, the observation of the German 
authors that the trypanosomes no longer develop in the tubes after 
the onset of cold weather is confirmed by the studies made here, 
as will be pointed out later. As to that of the Dudukalovs, it would 
be of interest to know if the round bodies first found were flagellated. 
If so, the Russian authors were dealing with a process altogether 
like that displayed by Trypanosoma americanum. The more 
leisurely development can be explained by the rather low temperature 
to which their cultures were subjected. It should also be noted 
that the figures accompanying their original article — the resum6 
given above having been taken from a review in a less difficult lan- 
guage than Russian — show that they were dealing with a trypanosome 
morphologically identical with T. americanum. On the other hand, 
it was not possible to get the American trypanosome to develop in 
salt solution, even though an equal bulk of blood had been added. 

Stockman (1910) found trypanosomes in the blood of 6 out of 
10 cattle which had been inoculated with Piroplasma to immunize 
against piroplasmosis. In one case the trypanosomes were present 
for eight days; in the other five for only a day or two. The try- 
panosomes were morphologically indistinguishable from T. theileri, 
and would not grow in culture tubes. 

Stockman makes no comment, but there is no reason for regarding 
what he found as anything but T. theileri, rendered more abundant 
as a result of the weakened condition of the cattle. 

METHOD OF THE EXPERIMENTAL WORK. 

The procedure followed was to prepare cultures of the blood of the 
cattle, and to examine fresh or fixed preparations made from these. 
The method of drawing the blood, making the cultures, etc., has 
been described in Bulletin 119, and need not be repeated here. The 
study falls easily into two parts, (1) that of the cultures themselves, 
or what may be called tlue experimental work, and (2) that made 
with the microscope on fresh or stained material. The work with 
the cultures will be detailed first. 

EXAMINATION OF CTJLTTJRES. 

In all, several hundred cultures were made. The media used 
were beef bouillon, mutton bouillon, bouillon made from extract of 
meat, and salt solution. In some cases the cultures were made the 



EXAMINATION OF CULTURES. 11 

same day as the blood was drawn; in others an interval of from 
one to several days was allowed to elapse, the blood meanwhile 
being kept either in the ice chest, in the laboratory, or in the incubator. 
Further, the blood was sometimes used in measured quantities; 
in other cases there was merely added to the medium what was 
considered to be a sufficient amount. 

At first the cultures were in some cases placed in the incubator, 
but the behavior of Typanosoma americanum is here the same as 
that of any other trypanosome in culture, and cultures kept at 
incubator temperature quickly deteriorate. The explanation has 
been advanced that the preference of a ' 'cultural" form for a moderate 
temperature is due to the fact that the biting arthropod which 
removes it from the mammalian host has the temperature of the 
surrounding air. Without dogmatizing as to whether or not this 
may be true, it is perfectly evident that the reason T. americanum 
can not live in cultures in the incubator is because these cultures 
spoil. Apparently, without reference to the presence or absence of 
bacteria, the hemoglobin leaches out of the red cells and goes into 
solution in the medium, and there can be no doubt that the 
hquids in the tube undergo profound chemical changes. Hence, 
in a very great majority of cases, the cultures were kept at room 
temperature, which varied from 20 to 27.5° C, according to the 
time of year. 

In the greater number of cultures the medium used was neutral 
beef bouillon, prepared in the laboratory. The tubes were charged 
the same day the blood was drawn, and the quantity of blood, 
while not measured, varied from 1 to 4 c. c. The quantity of bouillon 
was not measured accurately, but ranged from 3 to 6 c. c. It was 
mainly from such cultures as these that the material used in the 
microscopical studies was obtained. 

It is not necessary to tabulate the results obtained from this lot of 
cultures, as such. The more interesting results were obtained 
from those experiments in which the quantity of blood was measured 
and from those carried out with reference to the season of the year. 
The former are necessarily an entirely different set from those in 
which the blood was not measured, and the latter set was made 
up of both measured and unmeasured cultures. Tables for both of 
these sets of tubes are given later. 

It is evident that in order to determine the number of days required 
for the trypanosomes to appear in a tube, the tube must be examined 
daily from the time of making until the trypanosomes appear. 
This was done for 53 tubes, taken throughout the year, and it was 
found that the average time required was 3^ days. It is understood 
that this means the time required for the presence of trypanosomes 
on the top of the column of red cells to be determined or at least 



12 TRYPANOSOMA AMERICANUM, A BLOOD PARASITE OF CATTLE. 

suspected, merely by the use of a hand lens, although in almost 
all cases this diagnosis was confirmed by the use of the microscope. 
This average of 3^ days is based on cultures made the day the blood 
was drawn and which were kept at room temperature. 

In all 30 animals were used, and 64 separate tests were made of 
their blood. Of these animals 27 were yearlings or adults and 3 were 
young calves. The calves, one of which was but 1 day old, were all 
negative. The mother of the 1-day calf, tested at the same time, 
was positive, an indication that trypanosomes can not pass the 
placenta. 

Of the 27 adult animals, 7 gave negative results, hence 74 per cent 
were infected. The actual figure, however, is probably higher than 
this, since a single test may be negative merely by accident, and of 
these 7 cattle, 5 were tested but once. Moreover, as will be shown, 
the time of year must be taken into account. 

The following were six of the cattle which proved negative, with 
the month when the test was made; the seventh. No. 685, is dealt 
with below: Nos. 666 and 668, tested in March; No. 667, tested in 
April; No. 739, tested in May and also in October; No. 536, tested in 
July; and No. 738, tested in October. 

By October the trypanosomes have become much less abundant in 
the blood, and an animal negative in that month might readily have 
been positive in July or August. Nevertheless, certain cattle do not 
harbor the trypanosomes, as the following history of cow No. 685 
shows: 



Date. 



Number of 
cultures 
made. 



Result. 



1909 

July 13 

July 20 

Aug. 18 

Aug. 26 

Aug. 30 

Sept. 2 

Sept. 8 

Nov. 17 



5 All negative. 
4 Do. 

3 Do. 

Received in the jugular 
vein 20 c. c. of blood from 
cow No. 089, known to 
carry trypanosomes. 



All negative. 
Do. 
Do. 
Do. 



This is apparently a case of natural immunity. The inoculation 
might have failed, but the constant negative findings for five months, 
including the greater part of the hot season, show that the animal was 
able to resist natural infection. There was nothing in the history of 
this animal to differentiate it from any other, and it was in precisely 
the same environment as all of the other cattle at the Experiment 
Station. In fact, both it and No. 689, from which it was inoculated, 
were kept for a part of the summer in the same pen. 



EXAMINATION OF CULTURES. 



13 



SEASONAL VARIATION IN NUMBER OF TRYPANOSOMES IN BLOOD. 

The seasonal variation in the abundance of trypanosomes is very 
well shown by the history of cow No. 218, as follows: 

Appearance of trypanosomes in cultures from cow No. 218. 



No. of culture. 


Date. 


Quantity 
of blood. 


Time re- 
quired for 
trypanosomes 
to appear. 


Remarks. 


244 


Aug. IS 
...do 


Not meas- 
ured.! 
do 


4days 

5 days 

6 days 

4 days 

5 days 

do 

4 days 

3 days 

2 days 

do 




245 .... 




294 


Aug. 31 
...do 


do 

do 


Blood kept on ice 1 day. 
Do. 


295 


299 


Sept. 1 
Sept. 2 
Sept. 3 
Sept. 8 
...do 


do 

do 

do 

do 

do.... 


Blood kept on ice 2 days. 


303 


Blood kept on ice 3 days. 


309 


Blood kept on ice 4 days. 


330 




331 




334 


.do 


do 




420 


Nov. 17 
...do 


Ic. c 

2 c. c 


Negative 

do 




421 




422 


...do 


3 c. c 


do 




423 . . ". (...do 


4c. 


do 


Contaminated. 


424 


...do 


5 c. C 


do 




425 


...do 


6c.C 

7 c. c 

8c.c 

9c. c 


6 days 

Negative — 
6 davs 

ao 




42f) 


...do 




427 


...do 




428 


...do 




429 


...do 


10 c. c 


do 




430 . 


...do 


Ice 

3 c. c 


Negative — 
do 




438 


...do 




440 


...do 


5 c. c 

6 c. c 

7c.c 

8 c. c 

lOc.c 


6 days 

Negative 

todays 

Negative 

5 days 




441 


...do 




442. 


...do 




443 


...do 




445 


...do 











1 From 1 to 4 c. c, probably averaging 3 c. c. 

From this table we see that in August and September the cultures 
were all positive, and some showed trypanosomes as early as the 
second day. On the other hand, of the 17 cultures made in November, 
only 7 were positive, with 5 days as the minimum time. Moreover, 
while the average quantity of blood used in August and September 
was probably about 3 c. c, and in no case exceeded 4. c. c, the least 
quantity to develop trypanosomes in November was 5 c. c, and one 
each of the cultures containing 7 and 8 c. c, respectively, were negative. 

In the case of cow No. 697, from which many cultures were made, 
it was found that a larger proportion of tubes were positive, and 
trypanosomes appeared more quickly in spring and summer than 
they did in the autumn. 

The table following shows all the cases where the time of appearance 
of the trypanosomes was determined. It gives the number of cultures 
so determined, the maximum and minimum time required for the 
trypanosomes to appear, in days, and the average of the whole number. 
There is also given the mean temperature of each month, as obtained 
at the Washington, D. C, station of the United States Weather 
Bureau, and the mean temperature of the zoological laboratory. 



14i TRYPANOSOMA AMERICANUM, A BLOOD PARASITE OF CATTLE. 
Time of appearance of trypanosomes in laboratory cultures, by months. 



Month. 



Number 
of tubes. 



Time of appearance. 



Maximum. Minimum. Mean, 



Temperature. 



Washing- 
ton. 



Labora- 
tory. 



1909 

April 

May 

Jime 

July 

August 

September 

October 

November 

1910 

March 

April 



Days. 



Days. 



Days. 
3.3 
3.0 
3.0 
3.3 
4.4 
3.3 
7.2 
6.2 



4.1 
3.3 



"F. "C. 

54.2=12.3 
64.4=18.0 
73.4=23.0 

74.7=23.7 
73.0=22.8 
66.4=19.1 
53.2=11.8 
50.8=10.4 



51.2=10.7 
57.9=14.4 



°F. 'C. 

72.5=22.5 
74.3=23.5 
78.7=25.9 
81.5=27.5 
79.1=26.2 
75.2=24.0 
70.4=21.4 
71.7=22.1 



74.4=23.6 
74.1=23.4 



The reason why the laboratory temperature is always higher than 
that of the open air is doubtless obvious enough, although attention 
may be called to the fact that in hot weather the closing of the labora- 
tory windows at 4.30 p. m. prevents any such nightly fall of tempera- 
ture as is usual in the open. The temperature as recorded by the 
Weather Bureau, although taken in the city itself, may be assumed to 
correspond to that of the Experiment Station of the Bureau of 
Animal Industry, the home of the cattle. Accordingly, the one 
column shows the fluctuations of temperature to which the animals 
harboring the trypanosomes were subjected, the second that in which 
the tubes were kept. The former can hardly be supposed to have any 
influence, since a mammal maintains its own temperature without 
reference to that of the surrounding air. Therefore in making com- 
parisons between the rates of growth of the trypanosomes at different 
times of the year, it is the temperature of the laboratory which should 
be taken into account and not that of the open country. 

In the following, taken from the table given above, the months are 
in the first two cases grouped with reference to uniformity of tempera- 
ture and in the last with reference to the time required for the cultures 
to develop. 

1 . Variation in time of appearance of trypanosomes although monthly temperatures are 

closely uniform. 



Month. 



Labora- 
tory tem- 
perature. 



Mean 
time of 
appear- 
ance. 



AprO, 1909 

November, 1S09 
October, 1909.. 



°F. 
72.5 
71.7 
70.4 



Days. 



3.3 
6.2 
7.2 



EXAMINATION OF CULTURES. 15 

2. Uniformity in time of appearance of trypanosomes in spring months. 



Month. 



Labora- 
tory tem- 
perature. 



Mean 
time of 
appear- 
ance. 



March, 1910 
AprU, 1910. 
May, 1909.. 



'F. 

74.1 

74.4 
74.3 



Days. 



4.1 

3.3 
3.0 



3. Uniformity in time of appearance of trypanosomes although variation in monthly 

temperatures is considerable. 



Month. 




Mean 
time of 
appear- 
ance. 



April, 1909 

May, 1903 

July, 1909 

September, 1909 
April, 1910 



Days. 



3.3 
3.0 
3.3 
3.3 
3.3 



In the first group it is seen that although the temperatures are very 
close, the time required in one case is more than double that of 
another. In the second group the maximum difference in time is 
only 1.1 days, but here the comparison is between the three spring 
months. In the third, although the times are so near ahke that the 
difference is neghgible, there is a maximum difference of 9° F. in the 
temperature. 

In the statement below the entire time during which the study 
was being carried on is divided according to the three seasons — spring, 
summer, and autumn. The average time required for the cultures 
to develop during each season is computed, due weight being given 
to the number of cultures used, and the mean temperature, both of 
the open country and the laboratory, are given. 

Time of appearance of trypanosomes according to seasons. 





Time of 
appear- 
ance. 


Mean temperature. 


Season. 


Outside. 


In lab- 
oratory. 




Days. 
3.39 
3.86 
6.04 


66.9 
73.7 
56.8 


"F. 
73.8 


Summer . . 


79.8 




72.4 







There is seen here a considerable seasonal difference in the time 
required for the trypanosomes to appear in the tubes. This is taken 
to be an index to their abundance. The mode of examination of the 
cultures was throughout much the same, namely, a preliminar}^ 
observation of the top of the column of blood ceUs with a hand lens, 
followed, when necessary, by the examination of a drop of the culture 



16 TRYPANOSOMA AMERICANUM, A BLOOD PARASITE OF CATTLE. 



under the microscope. The latter was undertaken only when the 
former left the question as to the presence or absence of trypanosomes 
in doubt. 

As we shall see later, multiplication of the tiypanosomes begins as 
soon as the blood containing them is removed from the cow. It 
therefore seems reasonable to regard their discovery in the culture 
tubes by means of the procedure indicated above as evidence of 
greater abundance rather than a mere rapid multiplicative rate. 
Hence it is believed that these figures furnish satisfactory evidence to 
show that whereas the trypanosomes are less abundant in autumn 
than in spring or summer, this difference is independent of tempera- 
ture. The figure for spring is also lower than that for summer, but 
this difference is not large enough to warrant any conclusions. 

EFFECT ON THE TRYPANOSOMES OF KEEPING BLOOD BEFORE CULTURES 

WERE MADE. 

The following table gives the results obtained when the blood was 
kept one or more days before the cultures were made. The several 
columns show, in order, the number of the cow; date culture was 
made; date blood was drawn; interval in days; number of tubes used; 
number of tubes giving positive results; and place where blood was 
kept. 

Result of examination of laboratory cultures for trypanosomes. 



No. of cow. 



Culture 


Blood 


Interval 


Number 
of 


Number 
of posi- 


made. 


drawn. 


(days). 


tive 










cases. 


1909. 


1909. 








Apr. 2 


Mar. 31 


2 


3 





May 7 


May 2 


5 


5 





Aug. 19 


Aug. 17 


2 


4 





Aug. 27 


Aug. 26 


1 


2 





Aug. 27 


Aug. 26 


1 


2 





Aug. 28 


Aug. 26 


2 


2 





Aug. 28 


Aug. 26 


2 


2 





Aug. 29 


Aug. 26 


3 


2 





Aug. 29 


Aug. 26 


3 


2 





Aug. 30 


Aug. 26 


4 


2 





Aug. 30 


Aug. 26 


4 


2 





Aug. 31 


Aug. 26 


5 


2 





Aug. 31 


Aug. 30 


1 


2 





Aug. 31 


Aug. 30 


1 


2 


2 


Sept. 1 


Aug. 30 


2 


2 





Sept. 1 


Aug. 30 


2 


2 


1 


Sept. 2 


Aug. 30 


3 


2 





Sept. 2 


Aug. 30 


3 


2 


1 


Sept. 3 


Aug. 30 


4 


2 





Sept. 3 


Aug. 30 


4 


2 


1 


Sept. 4 


Sept. 3 


1 


2 





Sept. 4 


Sept. 3 


1 


2 


2 


Sept. 5 


Sept. 3 


2 


2 





Sept. 5 


Sept. 3 


2 


2 





Sept. 6 


Sept. 3 


2 


2 





Sept. 6 


Sept. 3 


3 


2 


1 


Sept. 14 


Sept. 13 


1 


2 





Sept. 15 


Sept. 13 


2 


2 





Sept. 16 


Sept. 13 


2 


2 





Nov. 4 


Nov. 3 


1 


4 


1 


Nov. 4 


Nov. 3 


1 


4 





Nov. 4 


Nov. 3 


1 


4 





1910. 


1910. 








Mar. 11 


Nov. 9 


2 


2 


2 


Mar. 29 


Nov. 16 


13 


4 






Place blood was 
kept. 



696. 
688 
689, 
218. 
689. 
218 
689 
218 
689. 
218. 
689, 
218. 
218. 
218. 
218. 
218. 
218. 
218. 
218. 
696. 
697. 
696. 
697 
696 
697 
218 
218 
218 
688. 
696 
697 

730 
730 



On ice. 
Do. 
In laboratory.' 

Do! 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 
In incubator. 
On ice. 
In incubator. 
On ice. 
In incubator. 
On ice. 
In incubator. 
On ice. 
In laboratory. 

Do. 

Do. 

Do. 

Do. 

Do. 
On ice. 

Do. 

Do. 
In Incubator. 

Do. 

Do. 



On ice. 
Do. 



EXAMINATION OF CULTURES, 



17 



The following data, taken from above, give the place where the 
blood was kept, the length of time kept, the total number of cultures 
made, and the number which developed tiypanosomes: 

Blood kept on ice. 



Time 


Culture 


Positive 


kept. 


tubes. 


cases. 


1 day... 


4 


2 


2 days... 


9 


3 


3 days... 


4 


1 


4 days... 


2 


1 


5 days. . . 


5 





13 days... 


4 






Blood kept in laboratory. 



Time 
kept. 


Culture 
tubes. 


Positive 
cases. 


Iday 

2 days.... 

3 days — 

4 days.... 

5 days — 


8 
12 
8 
8 
2 


2 

1 





Blood kept in incubator. 



Time 
kept. 


Culture 
tubes. 


Positive 
cases. 


Iday 

2 days.... 

3 days.... 

4 days.... 


14 
2 
2 
4 


1 






Thus, eleven cultures developed trypanosomes, and the following 
shows the time required in each case for the organisms to appear in 
the tubes: 

Blood on ice. 



Time kept. 


Time of 
appearance. 


Remarks. 


id^y--{b.-.".:::: 

(a 

2days...<b 

ic 

3 davs 


6 days 

3 days 

5 days 

4 days 

6-4 days... 

5 days 

4 days 


Good growth. 
Do. 
Do. 

Poor growth. 
Moderate growth. 






Mean 


4J days. . . . 



10414°— Bull. 145—12 3 



18 TRYPANOSOMA AMERICANUM, A BLOOD PARASITE OF CATTLE. 

Blood in laboratory. 



Time kept. 


Time of 
appearance. 


Remarks. 


iday.-ft::::::: 


3 days 

4-3 days... 


Very poor growth. 
Very poor growth; 

moderate growth. 
Not examined until 

15th day, when it 

was positive. 







Blood in incubator. 



Time kept. 


Time of 
appearance. 


Remarks. 


1 day.. 


12 days.... 


Very poor growth. 





When the blood was kept in the ice chest at a temperature of about 
16° C. for 1 to 5 days, only 7 out of 24 cultures developed, or 29 per 
cent. For blood kept in the laboratory the figure is 3 out of 38, or 8 
per cent. With blood kept in the incubator, but one culture out of 
22 produced trypanosomes. This shows that keeping the blood be- 
fore the cultures are made has a destructive influence on the trypano- 
somes. 

As to the time required for the trypanosomes to develop, the average 
is about 4^ days, yet tubes made from blood kept 4 days developed in 
4 days. Furthermore, whereas when the blood was kept in the labo- 
ratory only 2 out of 8 tubes developed, the trypanosomes appeared 
on the third and fourth days. The implication here is that the 
greater part of the trypanosomes in the blood are either killed or so 
enfeebled that they are unable to develop, while the growth which 
takes place is due to the hardier survivors. This is supported by the 
character of the growth which was eventually brought about in the 
tubes. In no case was this luxuriant, and it was good in only three 
of the tubes, pointing to the conclusion that the number of trypano- 
somes present in the tubes was very much below the average. 

In all of the cases considered above, control cultures made promptly 
after the blood was drawn were positive. 

RESULTS OBTAINED WITH MEASURED QUANTITIES OF BLOOD. 

The following table gives the results obtained with measured 
quantities of blood, the medium used being the laboratory make of 
beef bouillon, and the cultures being made the same day the blood 
was drawn. Unless otherwise stated, the culture tubes were kept in 
the room. In all cases given in the table where the quantity of blood 
was small, and the results negative, controls showed the blood used 
to contain trypanosomes. 



EXAMINATION OF CULTURES. 
Results obtained with measured quantities of blood. 



19 



Culture. 


Date. 


Cow. 


Quantity 

of 

blood. 


Result (+, 
positive;—, 
negative). 


Time. 


4 


1909 
Mar. 29 
...do 


688 
088 
688 
688 
688 
688 
688 
688 
088 
688 
688 
688 
688 
688 
688 
088 
695 
695 
695 
095 
695 
695 
696 
696 
696 
6% 
696 
696 
696 
696 
697 
697 
697 
697 
697 
697 
697 
697 
688 
688 
688 
688 
• 688 
688 
688 
688 
688 
688 
688 
688 
688 
688 
218 
218 
218 
218 
218 
218 
218 
218 
218 
218 
218 
218 
218 
218 
218 
218 
218 
218 

697 
697 
697 
697 
697 
697 


c.c. 
3.0 
1.5 
2.0 
1.5 
2.0 
2.5 
2.0 
1.5 
3.0 
5.0 
4.0 
4.0 
4.0 
4.0 
3.5 
3.0 
2.0 
1.5 
.4 
1.5 
3.5 
2.5 
3.0 
5.0 
3.5 
5.0 
4.0 
5.0 
3.0 
4.0 
2.5 
2.0 
2.0 
1.0 
2.5 
2.5 
1.0 
.5 
2.5 
3.0 
2.0 
2.0 
2.5 
2.0 
3.0 
2.0 
1.5 
1.5 
2.0 
2.5 
3.0 
3.0 
1.0 
2.0 
3.0 
4.0 
5.0 
6.0 
7.0 
8.0 
9.0 

10.0 
1.0 
3.0 
.5.0 
6.0 
7.0 
8.0 
9.0 

10.0 

Drops.' 
1 

2 

1 

2 

3 

4 


1 1 1 M 1 + 1 1 + ! t- 1 1 + + + 1 + 1 1 1 1 1 + + + + + + + + + + + + + + + + + + + + + + + + + 1 + 1 + + + + + + + + + + + + + + + + 1 ++ 1 ++ 1 + 


Days. 
'6 


5 


(') 
3 


31 


Apr. 5 
...do 


33 


4 


38 


...do.. 


3 


39 


Apr. 7 
...do.. 


41 


3 


42 


...do 


0) 
4 


43 


...do 


53 


Apr. 12 
...do 


3 


54 


4 


55 


..do.. .. 


3 


56 


...do 


3 


57 


..do. 


4 


58 


...do 


3 


59 


-do. 


3 


64 


\pT. 24 
.do. 


4 


65 




66 


..do 




67.... 


.do. 




68 


.do. 




70 


...do 




72 


.do. 




73 


...do 


4 


74 


.do. 




75 


.do 




76 


do. 




77 


.do. ... 




78 


..do 




79 ; 


do. 




81 


Apr. 26 
...do 


4 


82 




83 


.do 




85 


...do 




86 


.do. 




87 


.do 




88 






89 


.do. 




133 


May 13 
.do. 


3 


134 


3 


135 


...do 


3 


136 


.do. 




137 


.do. 




138 


do. 


3 


139 


.do. 


2 


140 


..do 


3 


141 


.do. 




142 


-do. 




144 


do. 


3 


145 


.do. 


3 


140 


do. 


3 


147 


.do. 


3 


420 


Nov. 17 
.do. 




421 




422 


do 




423 


.do. 




424 


..do. 




425 


-do. 


6 


426 


.do. 




427 


do. 


6 


428 


.do. 


6 


429 


do. 


6 


436 


.do. 




438 


do 




440 


.do. 




441 


do 




442 


.do. 




443 


do 




444 


do. 




445 


do. 


5 


532 


1910 
Apr. 7 




533 




550 


Apr. 13 




551 


.do. 




552 


do. 




5.53 


...do 





' Culture in incubator. 



» 1 drop=0.067o n. c. 



20 TRYPANOSOMA AMERICANUM, A BLOOD PARASITE OF CATTLE. 
Results obtained with measured quantities of blood — Continued. 



Culture. 



674. 
675. 
576. 
577. 
618. 

622. 
623. 
624. 
625. 



Date. 



1910 
Apr. 21 

..do 

..do 

..do 

July 14 



.do. 
.do. 
.do. 
.do. 



Cow. 



697 
697 
697 
697 
697 

697 
697 
697 
697 



Quantity 

of 

blood. 



Drops. 
5 
5 

10 

10 
5 
c. c. 
1.0 
1.0 
1.0 
1.0 



Result ( + , 
positive; — , 
negative). 



Days. 



Here it is seen that the smallest quantit}^ of blood to give a positive 
result was 5 drops, or 0.3375 c. c, and that this result was obtained 
only once out of three trials. Assuming 6,000,000 red cells and 10,000 
leucocytes per cubic millimeter, we find as a possible proportion one 
trypanosome for 2,022,000,000 red cells and 3,370,000 whites. Hence 
to find the trypanosome in the circulating blood would be merely a 
piece of good fortune. Further, culture 444, containing 9 c. c. of 
blood, was negative, yet this amount of blood contains 90,000,000 
leucocytes. 

For the most part the bouUlon used for the cultures was that made 
in the laboratory from beef, for ordinary bacteriological work. Some 
16 tubes were used in which the medium was made from commercial 
meat extract. It was not treated with an alkali, and hence was 
acid in reaction. The growth in the latter medium was in general 
not so luxuriant as that in the laboratory make of bouillon, and in 
two cases the trypanosomes failed to appear at all, although con- 
trols (in laboratory bouillon) were positive. 

Fourteen cultures were made in physiological salt solution, 6 parts 
per thousand. In one set of experiments with this medium 5 c. c. 
of salt solution was used in each tube, to which were added 2, 3, 4, 
and 5 c. c. of blood, respectively. Trypanosomes never developed, 
although in all cases the controls were positive. These results were 
at odds with those obtained by the Dudukalovs (see page 9). 

In one set of tubes cow's milk was used, the results being negative. 

GENERAL RESULTS OF CULTURAL WORK. 

• The results may be summarized as follows: 

(1) Trypanosoma aTnericanum grows readily in ordinary bouillon, 
made from either beef or mutton. 

(2) The average time required for the trypanosomes to be readily 
detected in the tubes is 3 J days. 



OBSERVATIONS ON FEESH MATERIAL. 21 

(3) The smallest quantity which gave positive results was 5 drops, 
or 0.3375 c. c. The largest measured quantity to give negative 
results was 9 c. c. 

(4) There is a seasonal fluctuation, the trypanosomes being more 
abundant m the spring and summer than they are in the autumn. 

OBSERVATIONS ON FRESH MATERIAL. 

The successful cultures were those made by adding the blood of 
the cow to bouillon of several different kinds. The freslily made 
culture necessarily consisted of a red column, composed of an admix- 
ture of blood and the medium. Upon standing for about 24 hours 
the blood cells settled to the bottom of the tube, leaving above them 
a clear fluid composed partly of blood serum and partly of bouillon 
itself. At first the surface of this column was smooth and of a 
uniform red, but by the end of the second day it usually began to 
present appearances which were presumably caused by the rising to 
the surface of the leucocytes. These appearances, however, were 
very varied. In some cases the surface remained perfectly flat but 
showed itself studded w^ith minute whitish specks and motes which, 
when large enough for their form to be determined, were of irreg- 
ular contours and closely resembled particles of dust. Frequently, 
however, a fuzzy deposit appeared upon the surface, consisting of a 
reddish-white stringy mass, which might be elevated as much as 
1 or 2 mm. These fuzzy masses never occupied more than a por- 
tion of the surface of the column of red cells. At other times this 
surface was differentiated into a series of ripples, representing in 
miniature the surface of wind-blown sand. 

But in spite of these various aspects, there was never any difli- 
culty in determining macroscopically (or preferably with the aid of 
a hand lens) whether the tubes did or did not contain trypanosomes, 
at least after they had had time, if present, to establish themselves. 
Frequently by the tliird day there could be distinguished on the 
surface of the red cells minute dots, differing from the motes above 
mentioned in that they were white and circular. When these had 
reached a certain size it could generally be seen that their top sur- 
faces were not flat but convex, and when as sometimes happened 
they were supported b}' the masses of fuzz and thus carried above 
the surface they showed as more or less spherical bodies. At first 
these trypanosome colonies were- very minute and frequently scat- 
tered over the surface in a more or less uniform manner, but as the 
culture got older the}' always became larger and often fewer in 
number. Increase in size is of course a matter of gi'owth, and the 
diminution in numbers was doubtless a case of fusion of closely 
lying colonies. In mature cultures there were at times but two or 
tliree colonies — enormous white masses 2 or 3 mm. across. 



22 TRYPANOSOMA AMERICANUM, A BLOOD PARASITE OF CATTLE. 

The small colonies were alwa3^s circular, the larger circular, oval, 
or irregular. A curved outline, however, was always maintained. 
The form of the colonies, however, is doubtless merely a matter of 
surface tension. Fusion of two very small circular colonies results 
in the production of a large circular one, but when a certain size is 
reached the surface tension is able only to impiart to the compound 
colony a curved outline. It was at times to be noted that the sep- 
arate units composing the large compound colonies were very incom- 
pletely fused. 

Clearly, each separate colony which arose on the surface of the 
red cells in a tube represented a focus of infection, the later increase 
in bulk being merely due to the multiplicative activity of the tryp- 
anosomes. No accurate counts were rnade, but such foci probably 
ranged in numbers, in the various tubes, from half a dozen or less to 
perhaps 100. These foci of infection must in each case be either a 
single trj^panosome, or some element not a trypanosome but which 
evolves into one, or two or more trypanosomes or such elements. 

In the preliminary notice (Bulletin 119) it was stated that it was 
not possible to say what this was, that is, whether the element in 
the circulating blood of the cow was an actual trypanosome or some- 
thing of an entirely different facies, and unfortunately such observa- 
tions as have been made since do not settle this point absolutely, 
yet they leave little doubt resting upon it. Trypanosomes were 
never seen in fresh blood from the cow, but they finally were found 
in stained smears of centrifuged blood, the smears having been made 
3 or 4 hours after the blood was drawn. It may be stated here that 
these organisms did not occur singly, but in clusters, and were 
evidently in a process of rapid multiplication. The probability that 
they arose from the division of a trypanosome, and not from some- 
thing else, is so great as to be a practical certainty. The work of 
Wrublewski and Behn, previously quoted, supports this hypothesis. 
In that of the former, the trypanosomes described as parasites of 
the aurochs are so like T. americanum that the latter might be 
regarded as little more than a variety of the former. The failure 
to find the trypanosome in the ])erfcctly fresh blood of American 
cattle is of little moment, since if the organism is only a variety of 
T. wniblewski it is in a strange host, and hence might be able to 
maintain itself in only very limited numbers. Moreover, it is a 
matter of common knowledge that trypanosomes may exist in the 
blood of animals in such small numbers as to evade observation with 
the microscope. A great many such cases have been brought out in 
the experimental work on the pathogenic trypanosomes of mammals, 
and it seems to be almost the rule for those of birds. Evidently, 
when a trypanosome can not be foinid in the blood with the micro- 
scope, it is more or less of a venture to claim that it exists there as 



OBSERVATIONS ON FRESH MATERIAL. 23 

a trypanosome and not as something else. Yet in the case of birds 
it is known that after faikire to find them in the circulating blood 
they have been picked up in extremely small numbers in the bone 
marrow. Besides, in spite of the enormous amount of work which 
has been done on these flagellates, nothing has ever been discovered 
in the blood but an element instantly recognizable as a trypanosome. 
Finally, there is Behn's discovery. But this trypanosome is very 
different from either T. americanum or T. wrublewsM. It is not only 
a very much broader animal, but differs in that the nuclei are farther 
back in the body. It is quite possible, however, that this tiypano- 
some, although the forerunner of the organisms found in the culture 
tubes by the German investigators, is a wholly different species 
from T. americanum. So far the cultural trypanosome of German 
cattle has neither been described nor figured, and it is hence impos- 
sible to compare it with that from American cattle. 

ATTEMPTS TO DISCOVER THE TRYPANOSOME IN FRESHLY DRAWN 

BLOOD. 

The experiments made at Washington to determme this point 
may now be given. 

Freshly drawn blood from cattle was centrifuged, and preparations 
were thereby obtained in which the leucocytes were as abundant as 
the red cells. This work was done in June, July, and August, 1910, 
when the cultural method showed the trypanosomes to be abundant. 
Six different animals were used, of which four were healthy, one was 
in an advanced stage of tuberculosis, and one was suffering from a 
mild case of Texas fever.^ 

A large number of fresh preparations were examined, but nothing 
in the way of a trypanosome was ever found. In a search of this 
sort the examination of fresh material was then and still is believed 
to be the most efficient method, since the flagellate readily betrays 
its presence by its motility. The work was done ^^^th a 16 mm. 
objective and a No. 12 eyepiece, and a preparation containing as 
much blood as a large smear could be searched in a few minutes. 
A thorough test of this procedure led to the view that the trypano- 
somes could not be found in recently drawn blood, and the examina- 
tion of stained smears was undertaken rather with the idea of looking 
for hypothetical ''preflagellate" stages than for actual trypanosomes. 
Yet it was in these that the discovery was made. The try^panosomes 
were found on two slides, a small group in each case, out of some 
25 examined. 

Cultures were also centrifuged. The first experiment, made July 
15, 1910, with a culture 23 to 24 hours old, revealed trypanosomes, 

1 This cow was tested in October. 



24 TRYPANOSOMA AMERICANUM, A BLOOD PARASITE OF CATTLE. 

singly or in clusters and either associated or not with white cells. 
Later experiments, made in August, reduccfl the time to 15 hours. 
Younger cultures were not tried. 

The findings, however, were of considerable interest. In the 
15-hour culture only a single trypanosome was found, but this was 
an organism having a body quite 20 microns in length, and a flagel- 
lum as long as the bod}', making it all of 40 microns long. The 
undulating membrane was short, and the appearance was typical 
for Typanosoma americanum. 

In a culture 18^ hours old made from blood 5^ hours after it was 
draA\Ti there were found several clusters of trypanosomes, one of 
which must have been composed of at least 75 individuals. Since in 
the culture tubes division of the flagellates is by no means a rapid 
process, requiring from 1 to 2 hours, multiplicative activity must 
have been going on for some time. At the slower rate, it would take 
14 hours for one trypanosome to produce 64. The organisms found 
on the first day in centrifuged cultures differed in no respect, so far as 
could be determined, from those found in two and three day cultures, 
studied without centrifuging. 

EVOLUTION OF THE TRYPANOSOMES IN CULTLTRE. 

The history of the evolution of the trypanosomes, as it takes place 
in a successful culture, may now be traced. 

As already stated, there is a certain variability in the rate of 
evolution, but it may conveniently be considered under tliree head- 
ings — the period of growth, of culmination, and of decline — although 
the three processes to a certain extent overlap. 

In cultures 2 to 3 days old there are present a small number of 
organisms, say from 1 to 6 to each preparation, a preparation 
probably containing 1 cubic millimeter of the culture. The animals 
are ordinarily short and relatively broad, with the anterior half 
broader than the posterior. At times, however, they are quite slender. 
A flagellum is always present and in most cases a short undulating 
membrane can easily be seen. The cytoplasm is hyaline. With 
ordinary light, granules can not as a rule be seen, but dark-field 
illumination shows them to be present. It may also here be noted 
that this method revealed the fact that in the adult trypanosomes 
the flagella often show a double contour, even to the extreme tip. 
The organisms are either solitary, in pairs, or in clusters. The 
clusters are nearly always of few individuals, but this of course 
depends upon the rate Avith which multiplicative activity has been 
inaugurated. The flagellum and undulating membrane are in 
constant action, and progression in a slow, misteady maimer, with 
numerous changes of direction, is frequent. 



OBSERVATIONS ON FRESH MATERIAL. 25 

Cultures 3 to 4 days old show all of the phenomena presented 
by the earlier cultures. There are very many more trypanosomes 
present, however, and the clusters average much larger. Solitary 
forms are relatively much rarer, almost all of the animals being 
in the clusters. The trypanosomes themselves have become longer. 
The flagella and undulating membranes are in constant motion, 
and it may here be stated that motility does not seem to fluctuate 
with age, senile and youthful forms showing it in approximately 
the same degree. 

Evolution now follows the lines already indicated. In the 4 
and 5 day cultures solitary forms occur, but are scarce. Nearly 
all the trypanosomes present are in clusters, which may consist of 
3 to 4 individuals, or be of enormous size. One, from a 4-day 
tube, measured 156 by 190 microns, and must have been composed 
of thousands of trypanosomes. The individual animals are larger; 
some long and slender, or crithidia-like ; others show a typical 
trypanosome contour, while still others have become club-shaped. 
Large, stout divisional forms are frequent. In many the cytoplasm 
has become very granular. 

From the fifth and sixth days onward the cultures begin to present 
a far more diversified appearance. There are present large and 
small clusters in which the trypanosomes may be band-shaped or 
club-shaped. There are many more free individuals, doubtless 
those which have become separated from the clusters. The free 
forms are band-shaped, club-shaped, or short oval bodies. Any and 
all of these may be in division. There also appear in the older 
cultures very large bodies with two or more flagella, no doubt result- 
ing from a more or less abnormal division process. The club-shaped 
forms and the giant trypanosomes are marks of degeneracy, and as 
the culture ages more and more of the trypanosomes begin to undergo 
senile decay, and the culture eventually dies out. 

No accurate data were kept as to how long the cultures generally 
lasted, but by the end of a month they are very evidently degenerate. 
In one case a tube charged 63 days previously was observed to 
show, microscopically, a large number of apparent colonies. A study 
of these colonies under the microscope showed, however, that they 
consisted merely of great masses of granular balls, with here and 
there a feebly motile, misshapen, and intensely granular trypanosome. 

The process of evolution of the individual, or of evolution com- 
bined with so-called involution, is not, except at the outset, difficult 
to follow. As given above, a solitary trypanosome having a total 
length of 40 microns was found in a centrifuged culture 15 hours old. 
(In the 2 and 3 day cultures the trypanosomes are not so large 
as this.) It will probably not be far from the mark to look upon a 
trypanosome of this size as the originator of the clusters which appear 



26 TRYPANOSOMA AMEEICANUM, A BLOOD PARASITE OF CATTLE. 

in the tubes. Division is always by longitudinal splitting, but may 
be ecjual or unequal. The observations rather favor the view that 
it is at first unequal, the original mother cell giving off a short, 
relatively broad bud. Such unequal divisions were seen in the 
ordinary (not centrifuged) cultures, and were not uncommon in one 
of the centrifuged cultures of the first day. At all events, beginning 
the series with a short, broad, flagellated element, the history is as 
follows: This element divides, and the two daughter cells are also 
at first short, broad organisms provided with flagella and undulating 
membranes. The cytoplasm is hyaline, and fine granules are prob- 
ably always present. An increase in length produces a relatively 
more slender form, which, in its turn, becomes a band-shaped 
typical trypanosome. In time, the anterior half becomes broader 
and rounded, like the head of a club, the posterior half remaining 
narrow. The flagellum extends forward from the anterior tip of 
the broad anterior half and carries with it a narrow band of 
cytoplasm, which is probably composed partly of endosarc, since 
it is frequently granular. The narrow posterior end becomes 
narrower; it is reduced to a mere spike, and finally disappears, 
leaving the monadine forms which show a flagellum either extending 
free or accompanied for a part of its length by a band of cytoplasm. 
In certainly a great many cases, however, and perhaps all, before 
the complete atrophy of the posterior end, the head of the club had 
undergone a distortion which carries the point of origin of the 
flagellum around to one side of the body, so that forms are obtained 
wherein the flagellum extends out at right angles to the longitudinal 
axis of the body. 

Along with the morphological changes there is a conspicuous 
change in the granularity of the cytoplasm. In the young trypano- 
somes the granules are small, but there is a gradual increase in size 
and numbers, and the senile forms are closely packed with coarse 
granules. The monadine form degenerates into a coarsely granular 
baU. 

The genesis of the colonies takes place as follows: A cluster is 
started, probably by a single trypanosome. This, by division, 
produces a pair, joined by their posterior ends. A second division 
produces a group of four, but the arrangement is not radial, but 
linear. The four are united, in common, by their posterior ends, 
but one of the pairs is slightly in advance of the other. In further 
divisions this linear arrangement is maintained, so that the clusters 
do not consist of rosettes, but of strings. Since, however, any given 
pair of trypanosomes is very nearly at the same level as the pair 
next to it, the linear disposition is more or less completely masked, 
and rounded clusters are produced. The actual arrangement may, 
however, be nicely demonstrated by observing a rounded cluster 



OBSERVATIONS ON FRESH MATERIAL. 27 

by dark-field illumination. As the cluster becomes heated by the 
rays it spreads out, becoming larger and looser, and then it can be 
easily seen that such a cluster consists of a number of closely related 
strings of trypanosomes. It may also be seen when trypanosomes 
have multiplied in a sealed preparation on a slide. Here, if the 
blood film be sufficiently thin, the groups are constrained to develop 
within one plane, and the linear arrangement is often very plain. 
Accordingly, the rounded cluster is a group of the second order, 
made up by the strings, wliichare in their turn made up of individual 
trypanosomes. Occasionally such strings (or perhaps a better 
term is bouquets) of trypanosomes were seen which were bipolar; 
that is, there were two strings extending from a central point in 
opposite directions. The strings may possibly also give off lateral 
branches. 

In view of the organization of the clusters, as just indicated, 
there is no objection to the view that each has arisen from a single 
trypanosome. If this be so, we should get, as a very rough average, 
say 25 to 50 trypanosomes per culture tube, or perhaps 10 per cubic 
centimeter of blood. This gives one trypanosome to 1,000,000 
white cells. The data given on page 20 show, however, that the 
trypanosomes are not so abundant as this. Such figures are little 
more than guesses, however, although they show that the trypano- 
somes are very rare in the circulating blood of the cow. . 

It may be of interest to give certain of the observations in detail: 
Fresh preparations from culture tubes 2 to 3 days old, examined 
under the microscope, show in abundance clumps composed partly 
of red cells and partly of leucocytes. These clumps stand out quite 
sharply from the film of red cells and evidently possess a considerable 
degree of consistency. Their production is probably in part due to 
the fact that as the leucocytes rise to the surface they tend to 
entangle a certain number of red cells; but there seems to be some 
other influence at work, because at times these clumps consisted 
entirely of red cells, and the red cells were always in the majority. 
In all cases the red cells, as a result of mutual pressure, were more 
or less misshapen. 

It was very quicklj^^ learned that the place to look for the first 
trypanosomes was in these clumps. In the early cultures one or two 
trypanosomes could usually be found in association with one of them, 
either in the midst of the blood cells or lying at the periphery, in the 
narrow clear space which usually separated a clump from the sur- 
rounding film of eiythrocytes. A day later there might be a con- 
siderable number of trypanosomes in such a situation, mostly in 
little clusters around the edges of the clump. In a number of cases, 
where preparations were obtained showing trypanosomes in associa- 
tion with the clumps, the position of these was determined by means 



28 TRYPANOSOMA AMERICANUM, A BLOOD PARASITE OF CATTLE. 

of the mechanical stage, and the preparation was set aside. Examina- 
tions on the following day always showed a conspicuous increase 
in the number of trypanosomes, and it was in such cases as these 
that the growth of the trypanosomes in strings or bouquets was at 
times demonstrated. 

The appearance of trypanosomes in association with these clumps 
of blood cells suggested at once that it was here that the origin of 
the foci of infection was to be found, and, since the clumps were 
small, that the problem of whether it was an actual trypanosome or 
some resting stage which gave birth to the trypanosomes in the 
cultures could easily be solved. In the endeavor to elucidate 
this point the dark-field illumination was at first used and some 
very deceptive appearances were noted. 

As stated above, these clumps are composed largely or wholly of 
red cells, the peripheries of which, with dark-field illumination, show 
as bright bands of a quite appreciable width, inclosing a space wliich 
does not reflect the Hght and is accordingly clear. Since the cells 
are closely packed together, the appearance is that of a region marked 
out with very irregular polygons. When the preparations studied 
were from cultures in an appropriate stage of development, that is, 
when the trypanosomes were still scarce, observation would at times 
appear to show the edges of one of these red cells to be in motion. 
Tliis motion at once recalled that of the flagellum of a trypanosome, 
as it indeed proved to be, for presently it could be seen that it was 
not the edge of the red cell that was in motion, but a thread, a flagel- 
lum, and by careful observation the flagellum could be traced to a 
faintly appearing trypanosome. The curious point about this phe- 
nomenon was, however, that although a point might be selected where 
the most careful scrutiny failed to reveal the presence of a trypano- 
some, the serpentine movement would presently manifest itself, and 
then the trypanosome itself could be discerned. It was as if the 
animals sprang into being full fledged. 

The mystery was solved by a study of the clumps by transmitted 
hght. It is probably a matter of common experience that fresh 
preparations are not very satisfactorily observed with an oil-immer- 
sion lens with daylight. The usual method was then somewhat 
modified. The small arc lamp belonging to the dark-field apparatus 
was used, its light bemg first passed through a piece of "euphos" 
glass, and then through a vessel containing a solution of methylene 
blue. Euphos glass cuts out the destructive ultra-violet rays; the 
methylene-blue solution is for the benefit of the observer's eyes, and 
can be made of whatever density desired. Finally — and this is of 
importance — immersion oil must be placed between the top of the 
condenser and the slide. This procedure permits the use of the 
highest-power eyepieces with a 2 mm. apochromatic lens, and the 



OBSEBYATIONS ON FRESH MATERIAL. 29 

tiypanosomes may be studied under powers of 1,500 to 2,250 
diameters. 

It was by this means learned that the trypanosomes which ap- 
peared so mysteriously were already present in the mass of red-blood 
cells, and had merely shifted their position. But, unfortunately, no 
light was shed on their origin. In a great many cases round cells, 
the appearance of which was not wholly that of normal leucocytes, 
were individually kept under observation for several hours, but 
nothing was ever seen to take place. The first trypanosomes ever 
seen in preparations from any culture were elongated, flagellated 
organisms. 

In the early cultures the trypanosomes occurred either singly or in 
groups of two or three. As has already been pointed out, the pairs 
arise by division, but there is another way whereby paired animals 
and clusters of very few might take origin. This would be by ag- 
glutination. Besides being associated with the clump of red and 
white cells, the trypanosomes are found wandering through the 
preparation. They create little or no disturbance amongst the red 
cells, since they travel at a slightly liigher level and may indeed be 
creeping on the under surface of the cover glass. Their movements 
are slow and unsteady and changes of direction are frequent. The fol- 
lowing detailed observations are typical of what may be seen: 

MOVEMENTS OF TRYPANOSOMES IN CULTURES. 

In a culture two days old, studied March 5, 1910, two young trypan- 
osomes came together at 4 p. m., and for 55 minutes what was seen 
can best be designated as a wrestling match between the two. Move- 
ments of the flagella and undulating membranes were practically 
continuous, and the two kept their bodies constantly curved, each 
looped around the other. At 4.55 they both straightened out and lay 
side by side, bodies in contact, the flagella pointing in the same direc- 
tion. Tliis position was, however, maintained but for a minute or 
two, after which they again bent their bodies, looped themselves 
together, and resumed the apparent struggle. At 5.30 the observa- 
tion was interrupted, to be resumed at 7.15 p. m., when the same 
condition of affairs was found. 

In another case, in a 3-day culture, two shghtly more mature 
flagellates were seen to come into contact and to remain so for several 
hours. Here, as in the case described above, the flagella and undu- 
lating membranes were in constant movement, and the animals also 
spent a part of the time each looped around the other. But, in 
addition, they would frequently partly separate and then each, with 
straight longitudinal axis, appose its body to that of the other. 
Wlien so disposed, they were oriented indifferently in the same or 



30 TRYPANOSOMA AMERICANUM, A BLOOD PARASITE OF CATTLE. 

opposite directions, and sometimes the end of one was opposite the 
middle of the body of the other. Under such circumstances it was 
generally quite impossible, even with the very favorable conditions 
under which the observations were made, to trace any line of demarca- 
tion between the two; but neither here nor in any other of the cases 
observed was there any reason to suspect conjugation. Neither 
is it believed that unions of this sort were permanent, since contact 
between the two was never by any specified portion of the body, and 
might be instantaneously broken, whereas the pairs arising by divi- 
sion remained attached, and always by their posterior ends. 

Wlien the trypanosomes were not rare in the mount, it was not at 
all unusual for a sohtary individual to leave the edges of the cell 
clumps and wander alone in the preparation. There might be quite 
a few trypanosomes wandering through the preparation, but there 
was never any evidence of any influence exerted at a distance. But 
in a number of cases it was noted that as soon as two individuals came 
into contact each at once became more energetic. 

THE PROCESS OF MLXTIPLICATION. 

The actual process of multiphcation was not seen many times, but 
the follo\ving olservation probably epitomizes the process: 

In a 4-day culture, at 2 p. m., March 7, 1910, there were found a 
large and a small trypanosome in contact. The former was a wholly 
normal animal of adult aspect, the latter egg-shaped but with a 
pointed posterior end and a short flagellum. It was rather less than 
half as long as the adult animal, and lay in contact with the anterior 
half of it. In a very few minutes it was seen that the flagellum of the 
egg-shaped element was much longer, and a little later the element 
itself was seen to be split halfway from the anterior to the posterior 
end. Unless the appearance of singleness of this body when first seen 
was deceptive, and due to the angle from which it was observed, the 
division into a partly separated element, with the two anterior ends 
each provided with a flagellum, did not require more than 15 or 20 
minutes. 

At 2.35 p. m. the large trj'panosome broke away from the paired 
element, but some 10 minutes later joined it again, wrapping its 
body around the pair in such a way that there was obtained an oval 
body provided with three flageUa at one end. The trio remained in 
intimate contact until 4.30 p. m., and during all of this time it was 
possible only momentarily to see an}" fine of demarcation between the 
three elements composing it. At 4.30 the large trypanosome again 
separated from the pair, and then it was possible to see that although 
the two small cells were still in contact by their posterior ends, they 
had increased very considerably in length, and were now much like 
the young forms seen in the young cultures. 



OBSERVATIONS ON FRESH MATERIAL. 31 

When the original pair was first seen it was taken to be a case of 
unequal division, but the subsequent behavior of the large trypano- 
some is rather against such an mterpretation. The little trjT)ano- 
somes, in which the body is oval, and may or may not be pointed at the 
ends, were frequently found, either singly or paired, in the clusters 
occurring in the young cultures, and other cases of division like that 
above described were at times seen. In these, so far as the observa- 
tions go, the pairs arising from division remained in contact, but it is 
not impossible that separation may sometimes take place before 
growth is completed, thus giving rise to sohtary oval forms. It is 
also theoretically possible that they arise from unequal division, 
as in Trypanosoma lewisi, but unfortunately neither my preserved 
material nor my observations on fresh material enable me to settle 
this point. 

Besides the oval form, there was occasionally found in the early 
cultures a round element, about as large as a red blood cell, provided 
with a long flagellum. Such are presumably to be regarded as modi- 
fications of the oval form. 

MOTILITY OF THE TRTPANOSOMES. 

Attention has been called to the almost constant motility of the 
trypanosomes. This was, however, subject to a certain variability, 
although it was not possible to determine upon what factors this 
variability depended. The normal movement was an undulation of 
the flagellum and undulating membrane, accompanied by a certain 
amount of flexion and torsion of the anterior part of the body. The 
younger trypanosomes were frequently seen to have their bodies 
bent into circles, simultaneously displaying energetic movements of 
the flagella and undulating membranes. Such positions might be 
maintained as long as the observation lasted. But in the fully 
fledged parasites the longitudinal axis was usually straight and the 
postnuclear part of the body rigid. 

At times trypanosomes, entangled amongst blood cells or granular 
debris, acted as though seeking to get free. In such cases periods of 
violent activity, during which the movements lacked all regularity, 
alternated with periods of complete rest. In the cases noted above, 
where two trypanosomes kept in company for considerable periods, 
the movements also lacked regularity. 

On one or two occasions, when the temperature of the laboratory 
during the night had been unusually low, the trypanosomes observed 
in the morning seemed rather sluggish. This observation suggested 
the foUowing experiment: A tube was placed in a vessel containing 
ice and salt, and kept at a temperature of just above freezing for 
nearly two hours. A preparation was then brought under the mi- 
croscope as quickly as possible, and immediately examined, with the 



32 TRYPANOSOMA AMEBICANUM, A BLOOD PARASITE OF CATTLE. 



result that the trypanosomes were found to be displaying an activity 
fully up to normal. 

Nor is it certain that heat increases motility. Trypanosomes 
always become much more active with dark-field illumination, but 
this may be laid to the ultra-violet or Roentgen rays rather than the 
heat. 

OBSERVATIONS ON FIXED MATERIAL. 

The observations made on fixed material confirm and supplement 
those made on fresh preparations. In one respect, moreover, they 

carry the study somewhat fur- 
ther, since in two cases smears 
made from fresh blood show 
trypanosomes (figs. 1 and 2) . 

The blood from wliich these 
smears were made was drawn 
at the Bureau Experiment Sta- 
tion, sent into the laboratory, 
and centrifuged before the 
preparations were made from 
it. The interval was probably 
about four hours. Hence the 
blood was not in a strict sense 
freshly drawn, yet the observa- 
tion compares with many others 
made upon trypanosomes. In 
many cases in the literature 
where trypanosomes are de- 
scribed as present in the cu-cu- 
lating blood, the blood in which 
they were found had either been 
removed from the living animal 
for some time or had even been 
taken from a dead animal. 
There had therefore been time 

¥iG.l.— 'Forms of Trypanosoma americanum horn. for it tO COol tO the tempcra- 
centrifuged blood. ^^^^ ^^ ^^^ ^-^.^ ^ ^^g^ • ^^ p^^^^^ 

is that of Trypanosoma wrvhlewski, found only in blood from dead 
bison. But in the case in hand it is believed that the removal of 
the trypanosomes from the circulation had an immediate effect, for 
the parasites, as seen in figures 1 and 2, are evidently in rapid 
division. Their extreme scarcity in the cow is practical proof that 
in such a situation the multiplicative energy is in abeyance. 

The only stimuli to which the parasites in the drawn blood were 
subjected were the lowering of the temperature, the defibrinating of 




OBSERVATIONS ON FIXED MATERIAL, 



33 



the blood, and the mechanical disturbance consequent upon cen- 
trifuging. It is hardly likely that defibrinating could have any effect, 
and the centrifuging was carried on for only a few minutes, the prepa- 
rations being then made at once. Hence no time was given for the 
number of divisions wliich the figures show to have taken place. 
Probably, then, it is the lowering of the temperature which induces 
multiplication, although it does not necessarily follow that this is in 
any way correlated with the removal of the trypanosomes by a biting 
fly. The mere cooling itseK might readily furnish all the stimulus 
needed. 



MORPHOLOGY OF THE FORMS IN THE BLOOD. 

Taking up now the trypanosomes as they appeared in the centri- 
fuged blood, it will be seen that they are normal in shape, flagellated, 
and possess a large tropho- 
nucleus and a conspicuous 
vacuole. Unfortunately, the 
stain used on the two slides 
on which they occurred was 
not in proper condition, and 
stained the cytoplasm so in- 
tensely that the situation of 
the kinetonuclei was ob- 
scured.^ 

For the group shown in 
figure 1 the average size of the 
body was 14.7 by 4 microns. 
In the case of figure 2 the 
dimensions were: Forms in 
division, 13.9 by 4 microns; 
forms not in division, 16.8 by 
3.6 microns. 

A selection of 14 of the fig- 
ured flagellates, confined to 
those wliich did not appear to 
have been greatly distorted 
in fixation, gave an average 

measurement of 16.8 by 3.8 microns. Newly born daughter cells 
can not be larger and are probably somewhat smafler than the 
mother cells from which they were derived. Hence the trypano- 
some, as it would appear in the circulating blood, probably has a 
length of at least 20 microns excluding the flagellum, and, as noted 
on page 24, a trypanosome of this size was found in a culture of the 
first dav. 




Fig. 2. — Forms of IWypanosoma Americanum from 
centrifuged blood. 



1 Efforts to improve the staining of these two slides were futile. The stain resisted a treatment with acid 
alcohol of sufQcient duration to injure the trypanosomes themselves. 



34 TRYPANOSOMA AMERIOANUM, A BLOOD PARASITE OF CATTLE. 
MORPHOLOGY OF THE FORMS IN CULTURE. 



Figures 3, 4, 5, and 6 show the appearance in smears from the 
early cultures. The shape is to a large extent artificial; for one 
thing, the early stages appear to contract on drying, becoming 
shorter and broader. Primarily, however, the distortion is due to 
the fact that as the spherical cluster dries it is fijKed flat, with results 
such as are shown very plainly in figure 5. The two cells shown in 
figure 4 are probably somewhat contracted. In the film they meas- 
ured 10 microns long. In figure 6 the largest trypanosome has the 
body 24.7 microns long. The group of six, shown in this figure, 
give clear indications of the method of their origin, and are proba- 
bly all derived from a single trypanosome. All but one are oriented 
in the same direction, and it is evident that this odd member of the 

group has just been forced out of the 
place which it had been occupying. 

Evolution, as already set forth on pages 
24 et seq., results in the production of 
longer and relatively and often actually 
more slender flagellates. Figure 7 from a 
culture on the fifth day shows two try- 
panosomes. They are longer and some- 
what more slender than those shown in 
figures 1 and 2. Figures 8 and 9 are from 
cultures of the same age. Here the ani- 
mals are long and slender, that of figure 8 
measuring 27.8 microns for the body. 
The undulating membrane also shows 
more distinctly in this figure. It was 
such forms as these which were spoken of 
as crithidial in the preliminary notice, 
but so much confusion exists in the literature as to what precisely 
are the characters of the genus Crithidia that the derived adjective 
is perhaps best let alone. 

Figures 11 to 15 show forms from older cultures. Evidently after 
a certain maximum length is reached the animals again become 
broader. Finally, by a considerable thickening of the anterior end, 
the club-shaped form is produced. Very marked, also, is the 
increased conspicuousness of the undulating membrane. 

In figures 13 and 14 are shown, among others, forms having a 
broad, rounded anterior end. Posteriorly the body is rigid and 
tongue-shaped, and this portion finally degenerates and disappears. 
Anteriorly the cytoplasm extends out in a long tapering portion, 
very flexible and mobile in the living animal and bearing the undu- 
lating membrane. This portion also becomes absorbed in the gen- 




FiG. 3.— Forms of Trypanosoma ameri- 
canum from bouillon culture 96 hours 
old. The individual members of 
the cluster are distinguished by the 
letters a to i. 



OBSERVATIONS ON FIXED MATERIAL.. 



36 



esis of the monadine forms, and trypanosomes having this outHne 
are doubtless more or less senile. 

Figure 12 shows the largest indi-vidual found in the preparations, 
the body alone measuring 38 microns. 

CYTOLOGY OF THE FORMS IN CULTURE. 

Along with the changes in size and shape there are conspicuous 
cytological changes. With reference to these, however, it is desir- 
able to say that the material used was all "dry," and all stained 







Figs. 4 to 9.— Forms of Trypanosoma americanum from bouillon cultures 112 hours old. 

with Wright's stain. Of late a good deal of the work on trypano- 
somes has been done with material fixed by the "wet" method, and 
the dry method has been extensively criticized. But the use of 
Wright's stain on dried blood is a standard procedure, and the results 
are so evidently accurate for such delicate structures as leucocytes 
that they can scarcely be very far wrong for trypanosomes. 

In the trypanosomes from the centrifuged blood of the cow the 
flagella stained, but poorly. In the early cultures, 2, 3, and 4 days, 
they stain, but often very faintly, this peculiarity being the cause of 
the error made in the preliminary notice. Later, however, their 



36 TRYPANOSOMA AMERICANUM, A BLOOD PARASITE OF CATTLE. 

aflinity for the stain becomes much greater, and in the fully fledged 
animals the flagella are conspicuous. The color is always red. In 
nearly all cases the tip is thickened to form a minute knob, as in 
Tryjjanosoma wrublewski. In the newly born forms the flagellum is 
short; in the adults it is long; probably on the average it is as long 
as the body. Frequently, however, in the older cultures, the flagel- 
lum is two or even three times longer than the body. 

In fresh preparations an undulating membrane is always in evi- 
dence, but in stained material made from young cultures it can not 
as a rule be made out. Later it broadens and becomes conspicuous, 
but is always short. 

The kinetonucleus from the first to the last stains intensely, 
usually a deep garnet color. It is sometimes round, more usually 
oval, and appears merely to constrict into two at the time of division. 
It seems always to be associated with a vacuole, which is at times 
as large as the trophonucleus. In cultures 2, 3, and 4 days old, 
when the trypanosomes are in rapid division, this vacuole is obvi- 
ously merely a cavity within the cytoplasm. 
It is of irregular outlines, sometimes lobu- 
lated, sometimes almost tubular. It presents 
the peculiarity that in these early stages of 
evolution it appears to be open to the exterior. 
The appearance presented recalls what is found 
in Euglena. Some euglenoid type might easily 
have been the ancestor of the Trypanosomidae, 
Fig. 10.— Trypanosoma ameri- in wliicli casc this vacuole might be regarded 

canwm from bouillon culture ,i i- ^ c j. i rnu i j. 

3 days old. as the rudimcnt ot a cytopharynx. 1 he data 

presented, however, are obviously insufiicient 
to warrant doing more than putting forward the above as a possible 
suggestion. 

Later, the vacuole becomes round or oval, ceases to give the 
appearance of being connected with the exterior, and furthermore 
absorbs the stain, becoming pink. Although in adult trypanosomes 
it frequently can not be demonstrated, it is perhaps a constant fea- 
ture and has been found in the monadine forms, the end terms of 
the series. 

As well as could be made out in the trypanosomes from the freshly 
drawn blood, the trophonucleus consisted of a large, tense sac, and 
such is clearly its condition in animals from the young cultures. It 
stains homogeneously and so faintly that the actual color is often 
diflicult to determine; generally is of a reddish cast. In the very 
early cultures — 2 and 3 days — the trophonucleus is very conspicu- 
ous, contrasting sharply with the blue cytoplasm. Later it becomes 
less homogeneous, presenting a ground substance in which is a 
quantity of amorphous matter having a different staining reaction. 




OBSERVATIONS ON FIXED MATERIAL. 



37 



In the late stages the appearance is that of the conventional tryp- 
anosome nucleus, an oval body provided with a number of large, 
rounded granules. The appearances, however, are manifold, and 
have not been worked out in detail. Presumably, two factors are 
at work — the age of the trypanosome and its state witli reference 
to division — and each of these is superposed upon the other. 







Figs. U and 12. — Forms of Trypanosoma americanum from bouil- 
lon cultures 6 days old. 
Figs. 13, 14, and 15. — Forms of Trypanosoma americanum from 
bouillon 10 days old. 

The cytoplasm in the early stages stains solidly, but is denser 
in the middle of the body than at the ends. It is always a clear blue. 
For the first several days it appears to be provided with a considerable 
number of small vacuoles of uniform size. Since, however, observa- 
tions on fresh material show these young trypanosomes to contain 
granules, and since there is no differentiation of the cytoplasm in the 
fixed preparations other than these apparent vacuoles, the probability 
is that they are really granules which refuse the stain. This sup- 
position receives support from the fact that the granules of leuco- 



38 TRYPANOSOMA AMERICANUM, A BLOOD PARASITE OF CATTLE. 

cytes at times appear as holes in the stained material. Later, the 
cytoplasm loses its soUdity and homogeneity and conspicuous blue 
and violet granules become abundant, and in old cultures the 
trypanosomes frequently consist of Httle else than sacs more or less 
completely filled with coarse granules. 

PRINCIPAL CHABACTEBISTICS OF TRYPANOSOMA AMERICANUM. 

Trypanosoma americanum is a large trypanosome. Figure 12 shows 
the largest specimen found, the body measuring some 38 microns in 
length. In this particular case the flagellum was either short or else 
failed to stain throughout. But since the flagellum is normally as 
long as the body, a total length of 75 microns is by no means unlikely. 

The most marked pecuUarity, however, is the very short undulating 
membrane. The kinetonucleus may be in front of, alongside, or 
behind the trophonucleus, and the two are always close together. 
But the reason for the shortness of the undulating membrane is that 
the nuclear system is pushed forward. The usual situation for the 
trophonucleus of a trypanosome is near the middle of the body, 
but in T. americanum it is at the junction of the anterior and middle 
thirds. That is, the distance from the middle part of the tropho- 
nucleus to the anterior end of the trypanosome averages 33 per cent 
of the body length, and in the specimens measured ranged from 
25 to 40 per cent. In this respect T. americanum differs from the 
T. transvaliense phase of T. theileri, in which, according to the data 
given by Luhs, this distance is about 50 per cent of the whole. Here 
again it agrees with T. wruhlewski, although even in the latter the 
nucleus, according to the published figures, is hardly so far forward. 

CONCLUSIONS. 

1. Trypanosoma americanum Hves, in all probability as a typical 
trypanosome, in the blood of perhaps 75 per cent of yearling and 
adult American cattle, but is not present in young calves. 

2. It comes very close to T wrublewsTci of the European bison, 
and may be only a variety of that species. 

3. Removal from the circulating blood stimulates multiplicative 
energy, apparently merely as the result of a cooler environment. 

4. Removed from the cow and placed under appropriate conditions, 
multiplicative energy runs far in advance of growth energy; hence — 

5. The trypanosomes divide and redivide very rapidly, and in 
consequence become smaller than the blood forms. 

6. At the end of a few days, multiplicative energy weakening, 
the organisms have an opportunity to grow and to reach their 
normal size. 

7. The adults are at first very slender, but in time increase in 
breadth and may become very large. 



CONCLUSIONS — BIBLIOGRAPHY. 39 

8. As the cultures reach and pass their maxima the individuals 
become club-shaped and eventually transform into rounded or 
oval elements, provided each with a long flagellum. 

9. Changes in the morphology of the nuclear system, and in the 
texture and chemical nature or composition of the cytoplasm, 
accompany changes in the facies of the entire organism. 

10. A distinguishing character is the situation of the trophonucleus, 
which is normally at the union of the anterior and middle thirds. 

BIBLIOGRAPHY. 

Behn, Paul. 

1910cr.— Uber Entwicklungsformen dea Trypanosoma Jranki <Berl. tierarztl. 

Wchnschr., v. 26 (42), 20. Oct., pp. 809-810, 1 fig. 
1910/3. — Priiflagellate Entwicklungsstadien der in deutschen Rindern kulturell 

nachweisbaren Trypanosomen <Berl. tierarztl. Wchnschr., v. 26 (46), 17. 

Nov., p. 899. 
1910;-. — Infektion eines Kalbes mit Trypanosomen vom Typus des Trypanosoma 

theileri mittelst Blut von Kiihen, in denen nur kulturell Flagellaten nachweis- 

bar waren <Berl. tierarztl. Wchnschr., v. 26 (50), 15. Dec, pp. 998-1000, figs. 

1—4. 

Delanoe, p. 

1911. — Presence de trypanosomes chez les bovid^ en France <Bull. Soc. path. 
exot., Par., v. 4 (2), 8 f^v., pp. 112-116. 

DUDUKALOV, A.; & DUDUKALOVA, N. 

1910. — Ob iskusstvennom kultivirovanii tripanozom naidennykh u rogatago 
skota. Sooshchenie vtoroe <Ark. vet. nauk, S.-Peterb., v. 40 (1), pp. 1-4. 
1 pi., figs. 1-16. ^ 

Knuth, Paul. 

1910. — Uber die in deutschen Rindern gefundenen Trypanosomen <Berl. tier- 
arztl. Wchnschr., v. 26 (42), 20. Oct., pp. 810-811. 

Knuth, Paul; & Rauchbaar, Gustav. 

1910. — Zum Vorkonunen von Trypanosomen bei Rindern in Deutschland. Zweite 
vorlilufige Mitteilung <Berl. tierarztl. Wchnschr., v. 26 (31), 4. Aug., pp. 
609-610. 

Knuth, Paul; Rauchbaar, Gustav; & Morgenstern, Paul. 

1910. — Nachweia von Trypanosomen beim Rinde im Kreise Oberwesterwald 
mittelst Zuchtung in Blut-Bouillon. Vorlaufige Mitteilung <Berl. tierarztl. 
Wchnschr., v. 26 (27), 7. Juli, pp. 539-540. 

Martini, Erich. 

1909. — The development of a piroplasma and trypanosoma of cattle in artificial 
culture media <Philippine J. So., Manila, v. 4 (3), June, pp. 147-169, pis. 1-6, 
fig. 1-34. 

MiYAJIMA, M. 

1907. — On the cultivation of a bovine piroplasma: A preliminary communication 
<Philippine J. Sc, Manila, B. Med Sc, v. 2 (2), May, pp. 83-92, pis. 1-3, 
figs. 1-12. 

Schmitt, F. M. 

1910. — Zum Vorkommen von Trypanosomen vom Typus der Trypanosoma theileri 
in deutschen Rindern <Berl. tierarztl. Wchnschr., v. 26 (44), 3. Nov., pp. 
841-842. 

Sergent, Edmond; & Etienne. 

1911. — Presence de trypanosomes chez les bovid^ en Algeria <Bull. Soc. path. 
exot.. Par., v. 4 (1), 11 Jan., pp. 40-42. 

Stockman, S. 

1910. — Preliminary note on a trypanosome of British cattle <J. Comp. Path. & 
Therap., Edinb. & Lond., v. 23 (2), June 30, pp. 189-192, 1 fig. 

o 



LB '12 



